PROJECT SUMMARY The discovery of specific molecular drivers of oncogenesis has led to a shift in the treatment of cancer patients, with a move away from the use of conventional cytotoxic chemotherapy and towards molecularly targeted agents that are often more effective and less toxic (e.g. EGFR and ALK inhibitors and immune- checkpoint inhibitor immunotherapies). However, this success of targeted inhibitors and immunotherapy has highlighted the challenge and importance of drug resistance. While patients often benefit from an initial and profound response to these current treatments, the vast majority of responses are incomplete and result in a residual disease state that serves as a prelude to subsequent tumor progression (acquired resistance). While several studies have delineated mechanisms of acquired resistance to treatment with various targeted inhibitors and immunotherapy, very little is known about the mechanisms underlying incomplete response and residual disease during initial treatment. This is a critical knowledge gap to fill to understand the longitudinal trajectories cancer cells take during treatment to form a drug-resistant tumor that leads to the clinical demise of patients. In Project 1, we will dissect the basis of incomplete response and resistance to targeted therapy and identify new treatment strategies to neutralize or eliminate residual disease and forestall resistance. We propose to do so through the prism of lung cancer, the foremost cause of cancer mortality worldwide and a paradigm-defining malignancy that illustrates both the successes and challenges of targeted therapy (and immunotherapy). A unique and transformative feature of our project is the ability to capture clinical specimens that include both liquid and tumor biopsies longitudinally from patients treated with targeted therapy (and immunotherapy), both early following treatment initiation and at maximal radiographic tumor response (residual disease). Coupled with our expertise in innovative methodologies such as tumor molecular profiling, genetic and pharmacologic screens, and organoid and patient-derived xenograft modeling, this capability to capture clinical samples from patients with residual disease affords an unprecedented window into the evolution of response and resistance in patients that can be leveraged to therapeutically target the residual disease state to enhance the magnitude and duration of response in patients. We will complement discovery efforts with the focused analysis of candidate modulators of residual disease that we have already uncovered. We propose 2 Specific Aims to characterize and therapeutically suppress residual disease during targeted therapy in NSCLC: Aim 1 will define the molecular portrait and identify therapeutic targets in targeted therapy residual disease in oncogene-driven non-small cell lung cancer (NSCLC). Aim 2 will functionally test the impact of target engagement to eliminate residual disease in oncogene-driven NSCLC patient-derived organoid and patient- derived xenograft (PDX) models.